Human pelota homolog

ABSTRACT

68772 polypeptides and polynucleotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing 68772 polypeptides and polynucleotides in the design of protocols for the treatment of proliferative diseases such as leukemias, solid tumor cancers and metastases; chronic inflammatory proliferative diseases such as psoriasis and rheumatoid arthritis; proliferative cardiovascular diseases such as restenosis; prolifertive ocular disorders such as diabetic retinopathy; and benign hyperproliferative diseases such as hemangiomas, among others, and diagnostic assays for such conditions.

This application is a division of U.S. application Ser. No. 09/145,947,filed Sep. 2, 1998, which is a division of U.S. application Ser. No.08/892,715, filed Jul. 15, 1997.

FIELD OF INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by them and to the use of such polynucleotides and polypeptides,and to their production. More particularly, the polynucleotides andpolypeptides of the present invention relate to Pelota family,hereinafter referred to as 68772. The invention also relates toinhibiting or activating the action of such polynucleotides andpolypeptides.

BACKGROUND OF THE INVENTION

Regulation of the cell cycle is controlled by a family of cyclins,cyclin dependent kinases (CDKs), CDK regulatory kinases, andphosphatases. See, Lees, E., Curr. Opin. Cell. Biol. 1995, 7:773-780;Piwinica-Worms, H., J. Lab. Clin. Med. 1996, 128:350-354. Progressionfrom the G2 phase to the M phase of the cell cycle requires the activityof cdc25 phosphatase. In Drosophila, mutations in the pelota gene havethe same phenotype as mutations in the twine and/or string genes, whichare cdc25 homologs, Eberhart, C. G. and Wasserman. S. A., Devel. 1995,121:3477-3486. Specific cell cycle effects of pelota mutations are seenin both meiosis and mitosis, including G2/M arrest between mitotic andmeiotic cell division, and disruption of nuclear envelope breakdown andspindle formation. Regulation of pelota offers a means of controlling acritical event in the cell cycle.

This indicates that the Pelota family has an established, proven historyas therapeutic targets. Clearly there is a need for identification andcharacterization of further members of Pelota family which can play arole in preventing, ameliorating or correcting dysfunctions or diseases,including, but not limited to, proliferative diseases such as leukemias,solid tumor cancers and metastases; chronic inflammatory proliferativediseases such as psoriasis and rheumatoid arthritis; proliferativecardiovascular diseases such as restenosis; prolifertive oculardisorders such as diabetic retinopathy; and benign hyperproliferativediseases such as hemangiomas.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to 68772 polypeptides andrecombinant materials and methods for their production. Another aspectof the invention relates to methods for using such 68772 polypeptidesand polynucleotides. Such uses include the treatment of proliferativediseases such as leukemias, solid tumor cancers and metastases; chronicinflammatory proliferative diseases such as psoriasis and rheumatoidarthritis; proliferative cardiovascular diseases such as restenosis;prolifertive ocular disorders such as diabetic retinopathy; and benignhyperproliferative diseases such as hemangiomas, among others. In stillanother aspect, the invention relates to methods to identify agonistsand antagonists using the materials provided by the invention, andtreating conditions associated with 68772 imbalance with the identifiedcompounds. Yet another aspect of the invention relates to diagnosticassays for detecting diseases associated with inappropriate 68772activity or levels.

DESCRIPTION OF THE INVENTION

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“68772 ” refers, among others, generally to a polypeptide having theamino acid sequence set forth in SEQ ID NO:2 or an allelic variantthereof.

“68772 activity or 68772 polypeptide activity” or “biological activityof the 68772 or 68772 polypeptide” refers to the metabolic orphysiologic function of said 68772 including similar activities orimproved activities or these activities with decreased undesirableside-effects. Also included are antigenic and immunogenic activities ofsaid 68772.

“68772 gene” refers to a polynucleotide having the nucleotide sequenceset forth in SEQ ID NO:1 or allelic variants thereof and/or theircomplements.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

“Isolated” means altered “by the hand of man” from the natural state. Ifan “isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not “isolated,” but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is “isolated”, as theterm is employed herein.

“Polynucleotide” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications has been made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short polynucleotides,often referred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from posttranslation natural processes ormay be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for protein modifications and nonproteincofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

“Variant” as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniquesor by direct synthesis.

“Identity” is a measure of the identity of nucleotide sequences or aminoacid sequences. In general, the sequences are aligned so that thehighest order match is obtained. “Identity” per se has an art-recognizedmeaning and can be calculated using published techniques. See, e.g.:(COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS,Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OFSEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, vonHeinje, G., Academic Press, 1987; and SEQUENCE ANALYSIS PRIMER,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).While there exist a number of methods to measure identity between twopolynucleotide or polypeptide sequences, the term “identity” is wellknown to skilled artisans (Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073). Methods commonly employed to determine identity orsimilarity between two sequences include, but are not limited to, thosedisclosed in Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J AppliedMath (1988) 48:1073. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and similarity between two sequences include, but arenot limited to, GCS program package (Devereux, J., et al., Nucleic AcidsResearch (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. etal., J Molec Biol (1990) 215:403).

As an illustration, by a polynucleotide having a nucleotide sequencehaving at least, for example, 95% “identity” to a reference nucleotidesequence of SEQ ID NO: 1 is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1. Inother words, to obtain a polynucleotide having a nucleotide sequence atleast 95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These mutations of the reference sequence may occur at the 5or 3 terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

Similarly, by a polypeptide having an amino acid sequence having atleast, for example, 95% “identity” to a reference amino acid sequence ofSEQ ID NO:2 is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of SEQ ID NO: 2. In other words,to obtain a polypeptide having an amino acid sequence at least 95%identical to a reference amino acid sequence, up to 5% of the amino acidresidues in the reference sequence may be deleted or substituted withanother amino acid, or a number of amino acids up to 5% of the totalamino acid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

Polypeptides of the Invention

In one aspect, the present invention relates to 68772 polypeptides (or68772 proteins). The 68772 polypeptides include the polypeptide of SEQID NO:2; as well as polypeptides comprising the amino acid sequence ofSEQ ID NO: 2; and polypeptides comprising the amino acid sequence whichhave at least 80% identity to that of SEQ ID NO:2 over its entirelength, and still more preferably at least 90% identity, and even stillmore preferably at least 95% identity to SEQ ID NO: 2. Furthermore,those with at least 97-99% are highly preferred. Also included within68772 polypeptides are polypeptides having the amino acid sequence whichhave at least 80% identity to the polypeptide having the amino acidsequence of SEQ ID NO:2 over its entire length, and still morepreferably at least 90% identity, and still more preferably at least 95%identity to SEQ ID NO:2. Furthermore, those with at least 97-99% arehighly preferred. Preferably 68772 polypeptide exhibit at least onebiological activity of 68772.

The 68772 polypeptides may be in the form of the “mature” protein or maybe a part of a larger protein such as a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

Fragments of the 68772 polypeptides are also included in the invention.A fragment is a polypeptide having an amino acid sequence that entirelyis the same as part, but not all, of the amino acid sequence of theaforementioned 68772 polypeptides. As with 68772 polypeptides, fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of 68772polypeptide. In this context “about” includes the particularly recitedranges larger or smaller by several, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes.

Preferred fragments include, for example, truncation polypeptides havingthe amino acid sequence of 68772 polypeptides, except for deletion of acontinuous series of residues that includes the amino terminus, or acontinuous series of residues that includes the carboxyl terminus ordeletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus Also preferred arefragments characterized by structural or functional attributes such asfragments that comprise alpha-helix and alpha-helix forming regions,beta-sheet and beta-sheet-forming regions, turn and turn-formingregions, coil and coil-forming regions, hydrophilic regions, hydrophobicregions, alpha amphipathic regions, beta amphipathic regions, flexibleregions, surface-forming regions, substrate binding region, and highantigenic index regions. Other preferred fragments are biologicallyactive fragments. Biologically active fragments are those that mediate68772 activity, including those with a similar activity or an improvedactivity, or with a decreased undesirable activity. Also included arethose that are antigenic or immunogenic in an animal, especially in ahuman.

Preferably, all of these polypeptide fragments retain the biologicalactivity of the 68772, including antigenic activity. Variants of thedefined sequence and fragments also form part of the present invention.Preferred variants are those that vary from the referents byconservative amino acid substitutions—i.e., those that substitute aresidue with another of like characteristics. Typical such substitutionsare among Ala, Val, Leu and Ile; among Ser and Thr; among the acidicresidues Asp and Glu; among Asn and Gln; and among the basic residuesLys and Arg; or aromatic residues Phe and Tyr. Particularly preferredare variants in which several, 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination.

The 68772 polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

Polynucleotides of the Invention

Another aspect of the invention relates to 68772 polynucleotides. 68772polynucleotides include isolated polynucleotides which encode the 68772polypeptides and fragments, and polynucleotides closely related thereto.More specifically, 68772 polynucleotide of the invention include apolynucleotide comprising the nucleotide sequence set forth in SEQ IDNO:1 encoding a 68772 polypeptide of SEQ ID NO: 2, and polynucleotidehaving the particular sequence of SEQ ID NO:1. 68772 polynucleotidesfurther include a polynucleotide comprising a nucleotide sequence thathas at least 80% identity over its entire length to a nucleotidesequence encoding the 68772 polypeptide of SEQ ID NO:2, and apolynucleotide comprising a nucleotide sequence that is at least 80%identical to that of SEQ ID NO:1 over its entire length. In this regard,polynucleotides at least 90% identical are particularly preferred, andthose with at least 95% are especially preferred. Furthermore those withat least 97% are highly preferred and those with at least 98-99% aremost highly preferred, with at least 99% being the most preferred. Alsoincluded under 68772 polynucleotides are a nucleotide sequence which hassufficient identity to a nucleotide sequence contained in SEQ ID NO:1 orcontained in the cDNA insert in the plasmid deposited with the ATCCDeposit number ATCC 98438 to hybridize under conditions useable foramplification or for use as a probe or marker. Moreover, 68772polynucleotide include a nucleotide sequence having at least 80%identity to a nucleotide sequence encoding the 68772 polypeptideexpressed by the cDNA insert deposited at the ATCC with Deposit NumberATCC 98438, and a nucleotide sequence comprising at least 15 contiguousnucleotides of such cDNA insert. In this regard, polynucleotides atleast 90% identical are particularly preferred, and those with at least95% are especially preferred. Furthermore, those with at least 97% arehighly preferred and those with at least 98-99% are most highlypreferred, with at least 99% being the most preferred. The inventionalso provides polynucleotides which are complementary to all the above68772 polynucleotides.

A deposit containing a human 68772 cDNA has been deposited with theAmerican Type Culture Collection (ATCC), 12301 Park Lawn Drive,Rockville, Md. 20852, USA, on May 28, 1997, and assigned ATCC DepositNumber ATCC 98438. The deposited material (clone) is SOLR containingUniZap (Stratagene, La Jolla, Calif.) that further contains the fulllength 68772 cDNA, referred to as “Human pelota cDNA clone from a humanT-cell library, ATG-1030” upon deposit. The cDNA insert is within EcoRI, Xho I site(s) in the vector. The nucleotide sequence of thepolynucleotides contained in the deposited material, as well as theamino acid sequence of the polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit has been made under the terms of the Budapest Treaty on theinternational recognition of the deposit of micro-organisms for purposesof patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

68772 of the invention is structurally related to other proteins of thePelota family, as shown by the results of sequencing the cDNA of Table 1(SEQ ID NO:1 ) encoding human 68772. The cDNA sequence of SEQ ID NO:1contains an open reading frame (nucleotide number 250 to 1407) encodinga polypeptide of 385 amino acids of SEQ ID NO:2. Amino acid sequence ofTable 2 (SEQ ID NO:2) has about 65% identity (using Gap in GCG(Needleman Wunsch)) in 395 amino acid residues with Drosophilamelanogaster pelota (Eberhart and Wasserman, Devel. 121:3477-3486,1995). Furthermore, 68772 (SEQ ID NO:2) is 36% identical toSaccharomyces cerevisiae DOM34 over 387 amino acid residues (Lalo etal., Compets Rendus de l'Academie des Sciences 316:367-373, 1993).Nucleotide sequence of Table 1 (SEQ ID NO:1) has about 63% identity(using Gap in GCG (Needleman Wunsch)) in 1186 nucleotide residues withDrosophila melanogaster pelota (Eberhart and Wasserman, Devel.121:3477-3486, 1995). Furthermore, 68772 (SEQ ID NO:1) is 45% identicalto Saccharomyces cerevisiae DOM34 over 1176 nucleotide base residues(Lalo et al., Compets Rendus de l'Academie des Sciences 316:367-373,1993) Thus 68772 polypeptides and polynucleotides of the presentinvention are expected to have, inter alia, similar biologicalfunctions/properties to their homologous polypeptides andpolynucleotides, and their utility is obvious to anyone skilled in theart.

TABLE 1^(a)CCCGGGCGCTGCAGTGTTCCCCGAGCCTGTTAGACGCAGCGCGCCGGGAGACTGAGAGAGGAAAGGATAGAGGAAGTGCTGCCCTAGGCTGCATGAGTCGAAGCAAGCGTGTTTCCTTCCCGCCAGGCAAGTGCCCTTAGAAACCGGGCCCCGCCCCCTTCCTGGCCTGCATTCCCATCCCCTCTCCCGGGGCGGAGGTGAGGACCTCCTTGGTTCCTTTGGTTCTGTCAGTGAGCCCCTTCCTTGGCCATGAAGCTCGTGAGGAAGAACATCGAGAAGGACAATGCGGGCCAGGTGACCCTGGTCCCCGAGGAGCCTGAGGACATGTGGCACACTTACAACCTCGTGCAGGTGGGCGACAGCCTGCGCGCCTCCACCATCCGCAAGGTACAGACAGAGTCCTCCACGGGCAGCGTGGGCAGCAACCGGGTCCGCACTACCCTCACTCTCTGCGTGGAGGCCATCGACTTCGACTCTCAAGCCTGCCAGCTGCGGGTTAAGGGGACCAACATCCAAGAGAATGAGTATGTCAAGATGGGGGCTTACCACACCATCGAGCTGGAGCCCAACCGCCAGTTCACCCTGGCCAAGAAGCAGTGGGATAGTGTGGTACTGGAGCGCATCGAGCAGGCCTGTGACCCAGCCTGGAGCGCTGATGTGGCGGCTGTGGTCATGCAGGAAGGCCTCGCCCATATCTGCTTAGTCACTCCCAGCATGACCCTCACTCGGGCCAAGGTGGAGGTGAACATCCCTAGGAAAAGGAAAGGCAATTGCTCTCAGCATGACCGGGCCTTGGAGCGGTTCTATGAACAGGTGGTCCAGGCTATCCAGCGCCACATACACTTTGATGTTGTAAAGTGCATCCTGGTGGCCAGCCCAGGATTTGTGAGGGAGCAGTTCTGCGACTACATGTTTCAACAAGCAGTGAAGACCGACAACAAACTGCTCCTGGAAAACCGGTCCAAATTTCTTCAGGTACATGCCTCCTCCGGACACAAGTACTCCCTGAAAGAGGCCCTTTGTGACCCTACTGTGGCTAGCCGCCTTTCAGACACTAAAGCTGCTGGGGAAGTCAAAGCCTTGGATGACTTCTATAAAATGTTACAGCATGAACCGGATCGAGCTTTCTATGGACTCAAGCAGGTGGAGAAGGCCAATGAAGCCATGGCAATTGACACATTGCTCATCAGCGATGAGCTCTTCAGGCATCAGGATGTAGCCACACGGAGCCGGTATGTGAGGCTGGTGGACAGTGTGAAAGAGAATGCAGGCACCGCTAGGATATTCTCTAGTCTTCACGTTTCTGGGGAACAGCTCAGCCAGTTGACTGGGGTAGCTGCCATTCTCCGCTTCCCTGTTCCCGAACTTTCTGACCAAGAGGGTGATTCCAGTTCTGAAGAGCATTAATGATTGAAACTTAAAATTGAGACAATCTTGTGTTTCCTAAACTGTTACAGTACATTTCTCAGCATCCTTGTGACAGAAAGCTGCAAGAAGGGCACTTTTTGATTCATACAGGGATTTCTTATGTCTTTGGCTACACTAGATATTTTGTGATTGGCAAGACATGTATTTAAACAATAAACTAAAAGGAAATAATCTCCACGTACTACCAAAAAAAAAAAAAAAAAA. ^(a)A nucleotide sequenceof a human 68772. SEQ ID NO: 1.

TABLE 2^(b)MKLVRKNIEKDNAGQVTLVPEEPEDMWHTYNLVQVGDSLRASTIRKVQTESSTGSVGSNRVRTTLTLCVEAIDFDSQACQLRVKGTNIQENEYVKMGAYHTIELEPNRQFTLAKKQWDSVVLERIEQACDPAWSADVAAVVMQEGLAHICLVTPSMTLTRAKVEVNIPRKRKGNCSQHDRALERFYEQVVQAIQRHIHFDVVKCILVASPGFVREQFCDYMFQQAVKTDNKLLLENRSKFLQVHASSGHKYSLKEALCDPTVASRLSDTKAAGEVKALDDFYKMLQHEPDRAFYGLKQVEKANEAMAIDTLLISDELFRHQDVATRSRYVRLVDSVKENAGTARIFSSLHVSGEQLSQLTGVAAILRFPVPELSDQEGDSSSEED. ^(b)An amino acid of sequenceof a human 68772. SEQ ID NO: 2.

One polynucleotide of the present invention encoding 68772 may beobtained using standard cloning and screening, from a cDNA libraryderived from mRNA in cells of human activated T-Cells, pancreas tumor,colon carcinoma, placenta, chondrosarcoma, hypoxic synoviocytes,osteroclastoma, tonsils, promyelocyte, heart, stimulated endothelialcells, and breast lymph node cells using the expressed sequence tag(EST) analysis (Adams, M. D., et al Science (1991) 252:1651-1656; Adams,M. D. et al., Nature, (1992) 355:632-634; Adams, M. D., et al., Nature(1995)377 Supp:3-174). Polynucleotides of the invention can also beobtained from natural sources such as genomic DNA libraries or can besynthesized using well known and commercially available techniques.

The nucleotide sequence encoding 68772 polypeptide of SEQ ID NO:2 may beidentical to the polypeptide encoding sequence contained in Table 1(nucleotide number 250 to 1407 of SEQ ID NO:1), or it may be a sequence,which as a result of the redundancy (degeneracy) of the genetic code,also encodes the polypeptide of SEQ ID NO:2.

When the polynucleotides of the invention are used for the recombinantproduction of 68772 polypeptide, the polynucleotide may include thecoding sequence for the mature polypeptide or a fragment thereof, byitself, the coding sequence for the mature polypeptide or fragment inreading frame with other coding sequences, such as those encoding aleader or secretory sequence, a pre-, or pro- or prepro-proteinsequence, or other fusion peptide portions. For example, a markersequence which facilitates purification of the fused polypeptide can beencoded. In certain preferred embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, as providedin the pQE vector (Qiagen, Inc.) and described in Gentz et al., ProcNatl Acad Sci USA. (1989) 86:821-824, or is an HA tag. Thepolynucleotide may also contain noncoding 5′ and 3 ′ sequences, such astranscribed, non-translated sequences, splicing and polyadenylationsignals, ribosome binding sites and sequences that stabilize mRNA.

Further preferred embodiments are polynucleotides encoding 68772variants comprise the amino acid sequence 68772 polypeptide of Table 2(SEQ ID NO:2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acidresidues are substituted, deleted or added, in any combination.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 80%, and preferably at least 90%, andmore preferably at least 95%, yet even more preferably 97-99% identitybetween the sequences.

Polynucleotides of the invention, which are identical or sufficientlyidentical to a nucleotide sequence contained in SEQ ID NO:1 or afragment thereof or to the cDNA insert in the plasmid deposited at theATCC with Deposit Number ATCC 98438 or a fragment thereof, may be usedas hybridization probes for cDNA and genomic DNA, to isolate fill-lengthcDNAs and genomic clones encoding 68772 polypeptide and to isolate cDNAand genomic clones of other genes that have a high sequence similarityto the 68772 gene. Such hybridization techniques are known to those ofskill in the art. Typically these nucleotide sequences are 80%identical, preferably 90% identical, more preferably 95% identical tothat of the referent. The probes generally will comprise at least 15nucleotides. Preferably, such probes will have at least 30 nucleotidesand may have at least 50 nucleotides. Particularly preferred probes willrange between 30 and 50 nucleotides.

In one embodiment, to obtain a polynucleotide encoding 68772 polypeptidecomprises the steps of screening an appropriate library under stingenthybridization conditions with a labeled probe having 15 the SEQ ID NO: 1or a fragment thereof; and isolating full-length cDNA and genomic clonescontaining said polynucleotide sequence. Thus in another aspect, 68772polynucleotides of the present invention further include a nucleotidesequence comprising a nucleotide sequence that hybridize under stringentcondition to a nucleotide sequence having SEQ ID NO: 1 or a fragmentthereof. Also included with 68772 polypeptides are polypeptidecomprising amino acid sequence encoded by nucleotide sequence obtainedby the above hybridization condition. Such hybridization techniques arewell known to those of skill in the art. Stringent hybridizationconditions are as defined above or, alternatively, conditions underovernight incubation at 42° C. in a solution comprising: 50 % formamide,5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate(pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 microgram/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65° C.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to animal and human disease.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, and hostcells which are genetically engineered with vectors of the invention andto the production of polypeptides of the invention by recombinanttechniques. Cell-free translation systems can also be employed toproduce such proteins using RNAs derived from the DNA constructs of thepresent invention.

For recombinant production, host cells can be genetically engineered toincorporate expression systems or portions thereof for polynucleotidesof the present invention. Introduction of polynucleotides into hostcells can be effected by methods described in many standard laboratorymanuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY(1986)and Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)such as calcium phosphate transfection, DEAE-dextan mediatedtransfection, transvection, microinjection, cationic lipid-mediatedtransfection, electroporation, transduction, scrape loading, ballisticintroduction or infection.

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, Streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanomacells; and plant cells.

A great variety of expression systems can be used. Such systems include,among others, chromosomal, episomal and virus-derived systems, e.g.,vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression systems may contain control regions that regulate as well asengender expression. Generally, any system or vector suitable tomaintain, propagate or express polynucleotides to produce a polypeptidein a host may be used. The appropriate nucleotide sequence may beinserted into an expression system by any of a variety of well-known androutine techniques, such as, for example, those set forth in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL (supra).

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the desired polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

If the 68772 polypeptide is to be expressed for use in screening assays,generally, it is preferred that the polypeptide be produced at thesurface of the cell. In this event, the cells may be harvested prior touse in the screening assay. It 68772 polypeptide is secreted into themedium, the medium can be recovered in order to recover and purify thepolypeptide; if produced intracellularly, the cells must first be lysedbefore the polypeptide is recovered. 68772 polypeptides can be recoveredand purified from recombinant cell cultures by well-known methodsincluding ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatographyis employed for purification. Well known techniques for refoldingproteins may be employed to regenerate active conformation when thepolypeptide is denatured during isolation and or purification.

Diagnostic Assays

This invention also relates to the use of 68772 polynucleotides for useas diagnostic reagents. Detection of a mutated form of 68772 geneassociated with a dysfunction will provide a diagnostic tool that canadd to or define a diagnosis of a disease or susceptibility to a diseasewhich results from under-expression, over-expression or alteredexpression of 68772. Individuals carrying mutations in the 68772 genemay be detected at the DNA level by a variety of techniques.

Nucleic acids for diagnosis may be obtained from a subject's cells, suchas from blood, urine, saliva, tissue biopsy or autopsy material. Thegenomic DNA may be used directly for detection or may be amplifiedenzymatically by using PCR or other amplification techniques prior toanalysis. RNA or cDNA may also be used in similar fashion. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to labeled 68772 nucleotide sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase digestion or by differences in melting temperatures.DNA sequence differences may also be detected by alterations inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents, or by direct DNA sequencing. See, e.g., Myers et al.,Science (1985) 230:1242. Sequence changes at specific locations may alsobe revealed by nuclease protection assays, such as RNase and S1protection or the chemical cleavage method. See Cotton et al., Proc NatlAcad Sci USA. (1985) 85: 4397-4401. In another embodiment an array ofoligonucleotides probes comprising 68772 nucleotide sequence orfragments thereof can be constructed to conduct efficient screening ofe.g., genetic mutations. Array technology methods are well known andhave general applicability and can be used to address a variety ofquestions in molecular genetics including gene expression, geneticlinkage, and genetic variability. (See for example: M. Chee et al.,Science, Vol 274, pp 610-613 (1996)).

The diagnostic assays offer a process for diagnosing or determining asusceptibility to proliferative diseases such as leukemias, solid tumorcancers and metastases; chronic inflammatory proliferative diseases suchas psoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas through detection of mutation in the 68772 gene by themethods described.

In addition, proliferative diseases such as leukemias, solid tumorcancers and metastases; chronic inflammatory proliferative diseases suchas psoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas, can be diagnosed by methods comprising determining from asample derived from a subject an abnormally decreased or increased levelof 68772 polypeptide or 68772 mRNA. Decreased or increased expressioncan be measured at the RNA level using any of the methods well known inthe art for the quantitation of polynucleotides, such as, for example,PCR, RT-PCR, RNase protection, Northern blotting and other hybridizationmethods. Assay techniques that can be used to determine levels of aprotein, such as an 68772 polypeptide, in a sample derived from a hostare well-known to those of skill in the art. Such assay methods includeradioimmunoassay, competitive-binding assays, Western Blot analysis andELISA assays.

Thus in another aspect, the present invention relates to a diagonostickit for a disease or suspectability to a disease, particularlyproliferative diseases such as leukemias, solid tumor cancers andmetastases; chronic inflammatory proliferative diseases such aspsoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas, which comprises:

(a) a 68772 polynucleotide, preferably the nucleotide sequence of SEQ IDNO: 1, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a 68772 polypeptide, preferably the polypeptide of SEQ ID NO: 2, ora fragment thereof; or

(d) an antibody to a 68772 polypeptide, preferably to the polypeptide ofSEQ D NO: 2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

Chromosome Assays

The nucleotide sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. The mapping of relevant sequences to chromosomes accordingto the present invention is an important first step in correlating thosesequences with gene associated disease. Once a sequence has been mappedto a precise chromosomal location, the physical position of the sequenceon the chromosome can be correlated with genetic map data. Such data arefound, for example, in V. McKusick, Mendelian Inheritance in Man(available on line through Johns Hopkins University Welch MedicalLibrary). The relationship between genes and diseases that have beenmapped to the same chromosomal region are then identified throughlinkage analysis (coinheritance of physically adjacent genes).

The differences in the cDNA or genomic sequence between affected andunaffected individuals can also be determined. If a mutation is observedin some or all of the affected individuals but not in any normalindividuals, then the mutation is likely to be the causative agent ofthe disease.

Antibodies

The polypeptides of the invention or their fragments or analogs thereof,or cells expressing them can also be used as immunogens to produceantibodies immunospecific for the 68772 polypeptides. The term“immunospecific” means that the antibodies have substantially greateraffinity for the polypeptides of the invention than their affinity forother related polypeptides in the prior art.

Antibodies generated against the 68772 polypeptides can be obtained byadministering the polypeptides or epitope-bearing fragments, analogs orcells to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature (1975) 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today (1983) 4:72) andthe EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES ANDCANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can also be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms including other mammals, may be used to express humanizedantibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides byaffinity chromatography.

Antibodies against 68772 polypeptides may also be employed to treatproliferative diseases such as leukemias, solid tumor cancers andmetastases; chronic inflammatory proliferative diseases such aspsoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas, among others.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in a mammal which comprises inoculating themammal with 68772 polypeptide, or a fragment thereof, adequate toproduce antibody and/or T cell immune response to protect said animalfrom proliferative diseases such as leukemias, solid tumor cancers andmetatases; chronic inflammatory proliferative diseases such as psoriasisand rheumatoid arthritis; proliferative cardiovascular diseases such asrestenosis; prolifertive ocular disorders such as diabetic retinopathy;and benign hyperproliferative diseases such as hemangiomas, amongothers. Yet another aspect of the invention relates to a method ofinducing immunological response in a mammal which comprises, delivering68772 polypeptide via a vector directing expression of 68772polynucleotide in vivo in order to induce such an immunological responseto produce antibody to protect said animal from diseases.

Further aspect of the invention relates to an immunological/vaccineformulation (composition) which, when introduced into a mammalian host,induces an immunological response in that mammal to a 68772 polypeptidewherein the composition comprises a 68772 polypeptide or 68772 gene. Thevaccine formulation may further comprise a suitable carrier. Since 68772polypeptide may be broken down in the stomach, it is preferablyadministered parenterally (including subcutaneous, intramuscular,intravenous, intradermal etc. injection). Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

Screening Assays

The 68772 polypeptide of the present invention may be employed in ascreening process for compounds which activate (agonists) or inhibitactivation of (antagonists, or otherwise called inhibitors) the 68772polypeptide of the present invention. Thus, polypeptides of theinvention may also be used to assess identify agonist or antagonistsfrom, for example, cells, cell-free preparations, chemical libraries,and natural product mixtures. These agonists or antagonists may benatural or modified substrates, ligands, receptors, enzymes, etc., asthe case may be, of the polypeptide of the present invention; or may bestructural or functional mimetics of the polypeptide of the presentinvention. See Coligan et al., Current Protocols in Immunology1(2):Chapter 5 (1991).

68772 polypeptides are responsible for many biological functions,including many pathologies. Accordingly, it is desirous to findcompounds and drugs which stimulate 68772 polypeptide on the one handand which can inhibit the function of 68772 polypeptide on the otherhand. In general, agonists are employed for therapeutic and prophylacticpurposes for such conditions as proliferative diseases such asleukemias, solid tumor cancers and metastases; chronic inflammatoryproliferative diseases such as psoriasis and rheumatoid arthritis;proliferative cardiovascular diseases such as restenosis; prolifertiveocular disorders such as diabetic retinopathy; and benignhyperproliferative diseases such as hemangiomas. Antagonists may beemployed for a variety of therapeutic and prophylactic purposes for suchconditions as proliferative diseases such as leukemias, solid tumorcancers and metastases; chronic inflammatory proliferative diseases suchas psoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas.

In general, such screening procedures may involve using appropriatecells which express the 68772 polypeptide or respond to 68772polypeptide of the present invention. Such cells include cells frommammals, yeast Drosophila or E. coli. Cells which express the 68772polypeptide (or cell membrane containing the expressed polypeptide) orrespond to 68772 polypeptide are then contacted with a test compound toobserve binding, or stimulation or inhibition of a functional response.The ability of the cells which were contacted with the candidatecompounds is compared with the same cells which were not contacted for68772 activity.

The assays may simply test binding of a candidate compound whereinadherence to the cells bearing the 68772 polypeptide is detected bymeans of a label directly or indirectly associated with the candidatecompound or in an assay involving competition with a labeled competitor.Further, these assays may test whether the candidate compound results ina signal generated by activation of the 68772 polypeptide, usingdetection systems appropriate to the cells bearing the 68772polypeptide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed.

The 68772 cDNA, protein and antibodies to the protein may also be usedto configure assays for detecting the effect of added compounds on theproduction of 68772 mRNA and protein in cells. For example, an ELISA maybe constructed for measuring secreted or cell associated levels of 68772protein using monoclonal and polyclonal antibodies by standard methodsknown in the art, and this can be used to discover agents which mayinhibit or enhance the production of 68772 (also called antagonist oragonist, respectively) from suitably manipulated cells or tissues.Standard methods for conducting screening assays are well understood inthe art.

The 68772 protein may be used to identify membrane bound or solublereceptors, if any, through standard receptor binding techniques known inthe art. These include, but are not limited to, ligand binding andcrosslinking assays in which the 68772 is labeled with a radioactiveisotope (eg 125I), chemically modified (eg biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (cells, cell membranes, cellsupernatants, tissue extracts, bodily fluids). Other methods includebiophysical techniques such as surface plasmon resonance andspectroscopy. In addition to being used for purification and cloning ofthe receptor, these binding assays can be used to identify agonists andantagonists of 68772 which compete with the binding of 68772 to itsreceptors, if any. Standard methods for conducting screening assays arewell understood in the art.

Examples of potential 68772 polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, enzymes, etc., as thecase may be, of the 68772 polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

Thus in another aspect, the present invention relates to a screening kitfor identifying agonists, antagonists, ligands, receptors, substrates,enzymes, etc. for 68772 polypeptides; or compounds which decrease orenhance the production of 68772 polypeptides, which comprises:

(a) a 68772 polypeptide, preferably that of SEQ ID NO:2;

(b) a recombinant cell expressing a 68772 polypeptide, preferably thatof SEQ ID NO:2;

(c) a cell membrane expressing a 68772 polypeptide; preferably that ofSEQ ID NO: 2; or

(d) antibody to a 68772 polypeptide, preferably that of SEQ ID NO: 2.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

Prophylactic and Therapeutic Methods

This invention provides methods of treating abnormal conditions such as,proliferative diseases such as leukemias, solid tumor cancers andmetastases; chronic inflammatory proliferative diseases such aspsoriasis and rheumatoid arthritis; proliferative cardiovasculardiseases such as restenosis; prolifertive ocular disorders such asdiabetic retinopathy; and benign hyperproliferative diseases such ashemangiomas, related to both an excess of and insufficient amounts of68772 polypeptide activity.

If the activity of 68772 polypeptide is in excess, several approachesare available. One approach comprises administering to a subject aninhibitor compound (antagonist) as hereinabove described along with apharmaceutically acceptable carrier in an amount effective to inhibitthe function of the 68772 polypeptide, such as, for example, by blockingthe binding of ligands, substrates, enzymes, receptors, etc., or byinhibiting a second signal, and thereby alleviating the abnormalcondition. In another approach, soluble forms of 68772 polypeptidesstill capable of binding the ligand, substrate, enzymes, receptors, etc.in competition with endogenous 68772 polypeptide may be administered.Typical embodiments of such competitors comprise fragments of the 68772polypeptide.

In still another approach, expression of the gene encoding endogenous68772 polypeptide can be inhibited using expression blocking techniques.Known such techniques involve the use of antisense sequences, eitherinternally generated or separately administered. See, for example,O'Connor, J Neurochem (1991) 56:560 in Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Alternatively, oligonucleotides which form triple helices withthe gene can be supplied. See, for example, Lee et al., Nucleic AcidsRes (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al.,Science (1991) 251:1360. These oligomers can be administered per se orthe relevant oligomers can be expressed in vivo.

For treating abnormal conditions related to an under-expression of 68772and its activity, several approaches are also available. One approachcomprises administering to a subject a therapeutically effective amountof a compound which activates 68772 polypeptide, i.e., an agonist asdescribed above, in combination with a pharmaceutically acceptablecarrier, to thereby alleviate the abnormal condition. Alternatively,gene therapy may be employed to effect the endogenous production of68772 by the relevant cells in the subject. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct may then be isolated and introduced intoa packaging cell transduced with a retroviral plasmid vector containingRNA encoding a polypeptide of the present invention such that thepackaging cell now produces infectious viral particles containing thegene of interest. These producer cells may be administered to a subjectfor engineering cells in vivo and expression of the polypeptide in vivo.For overview of gene therapy, see Chapter 20, Gene Therapy and otherMolecular Genetic-based Therapeutic Approaches, (and references citedtherein) in Human Molecular Genetics, T Strachan and A P Read, BIOSScientific Publishers Ltd (1996). Another approach is to administertherapeutic amount of 68772 polypeptides in combination with a suitablepharmaceutical carrier.

Formulation and Administration

Peptides, such as the soluble form of 68772 polypeptides, and agonistsand antagonist peptides or small molecules, may be formulated incombination with a suitable pharmaceutical carrier. Such formulationscomprise a therapeutically effective amount of the polypeptide orcompound, and a pharmaceutically acceptable carrier or excipient. Suchcarriers include but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof.Formulation should suit the mode of administration, and is well withinthe skill of the art. The invention further relates to pharmaceuticalpacks and kits comprising one or more containers filled with one or moreof the ingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

Preferred forms of systemic administration of the pharmaceuticalcompositions include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if properly formulated in enteric or encapsulatedformulations, oral administration may also be possible. Administrationof these compounds may also be topical and/or localized, in the form ofsalves, pastes, gels and the like.

The dosage range required depends on the choice of peptide, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.Wide variations in the needed dosage, however, are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladminstration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

Polypeptides used in treatment can also be generated endogenously in thesubject, in treatment modalities often referred to as “gene therapy” asdescribed above. Thus, for example, cells from a subject may beengineered with a polynucleotide, such as a DNA or RNA, to encode apolypeptide ex vivo, and for example, by the use of a retroviral plasmidvector. The cells are then introduced into the subject.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples illustrate, but do not limitthe invention.

Example 1

The full-length clone (68772) was identified through searches of theHuman Genome Sciences database.

Northern blotting of multiple tissue human RNA blots performed using68772 (human pelota) as a probe, detected a message of approximately 1.9kb in several cancer cell lines: HL-60, HelaS3, K-562, MOLT-4, Raji,SW480, A549, and G361. The message was also found in fetal liver,peripheral blood lymphocytes, and also weakly expressed in bone marrowand thymus. No apparent message was detected in brain, spleen, appendix,lymph node, heart, placenta, lung, liver, skeletal muscle, kidney, orpancreas.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

2 1632 base pairs nucleic acid single linear cDNA 1 CCCGGGCGCTGCAGTGTTCC CCGAGCCTGT TAGACGCAGC GCGCCGGGAG ACTGAGAGAG 60 GAAAGGATAGAGGAAGTGCT GCCCTAGGCT GCATGAGTCG AAGCAAGCGT GTTTCCTTCC 120 CGCCAGGCAAGTGCCCTTAG AAACCGGGCC CCGCCCCCTT CCTGGCCTGC ATTCCCATCC 180 CCTCTCCCGGGGCGGAGGTG AGGACCTCCT TGGTTCCTTT GGTTCTGTCA GTGAGCCCCT 240 TCCTTGGCCATGAAGCTCGT GAGGAAGAAC ATCGAGAAGG ACAATGCGGG CCAGGTGACC 300 CTGGTCCCCGAGGAGCCTGA GGACATGTGG CACACTTACA ACCTCGTGCA GGTGGGCGAC 360 AGCCTGCGCGCCTCCACCAT CCGCAAGGTA CAGACAGAGT CCTCCACGGG CAGCGTGGGC 420 AGCAACCGGGTCCGCACTAC CCTCACTCTC TGCGTGGAGG CCATCGACTT CGACTCTCAA 480 GCCTGCCAGCTGCGGGTTAA GGGGACCAAC ATCCAAGAGA ATGAGTATGT CAAGATGGGG 540 GCTTACCACACCATCGAGCT GGAGCCCAAC CGCCAGTTCA CCCTGGCCAA GAAGCAGTGG 600 GATAGTGTGGTACTGGAGCG CATCGAGCAG GCCTGTGACC CAGCCTGGAG CGCTGATGTG 660 GCGGCTGTGGTCATGCAGGA AGGCCTCGCC CATATCTGCT TAGTCACTCC CAGCATGACC 720 CTCACTCGGGCCAAGGTGGA GGTGAACATC CCTAGGAAAA GGAAAGGCAA TTGCTCTCAG 780 CATGACCGGGCCTTGGAGCG GTTCTATGAA CAGGTGGTCC AGGCTATCCA GCGCCACATA 840 CACTTTGATGTTGTAAAGTG CATCCTGGTG GCCAGCCCAG GATTTGTGAG GGAGCAGTTC 900 TGCGACTACATGTTTCAACA AGCAGTGAAG ACCGACAACA AACTGCTCCT GGAAAACCGG 960 TCCAAATTTCTTCAGGTACA TGCCTCCTCC GGACACAAGT ACTCCCTGAA AGAGGCCCTT 1020 TGTGACCCTACTGTGGCTAG CCGCCTTTCA GACACTAAAG CTGCTGGGGA AGTCAAAGCC 1080 TTGGATGACTTCTATAAAAT GTTACAGCAT GAACCGGATC GAGCTTTCTA TGGACTCAAG 1140 CAGGTGGAGAAGGCCAATGA AGCCATGGCA ATTGACACAT TGCTCATCAG CGATGAGCTC 1200 TTCAGGCATCAGGATGTAGC CACACGGAGC CGGTATGTGA GGCTGGTGGA CAGTGTGAAA 1260 GAGAATGCAGGCACCGCTAG GATATTCTCT AGTCTTCACG TTTCTGGGGA ACAGCTCAGC 1320 CAGTTGACTGGGGTAGCTGC CATTCTCCGC TTCCCTGTTC CCGAACTTTC TGACCAAGAG 1380 GGTGATTCCAGTTCTGAAGA GGATTAATGA TTGAAACTTA AAATTGAGAC AATCTTGTGT 1440 TTCCTAAACTGTTACAGTAC ATTTCTCAGC ATCCTTGTGA CAGAAAGCTG CAAGAAGGGC 1500 ACTTTTTGATTCATACAGGG ATTTCTTATG TCTTTGGCTA CACTAGATAT TTTGTGATTG 1560 GCAAGACATGTATTTAAACA ATAAACTAAA AGGAAATAAT CTCCACGTAC TACCAAAAAA 1620 AAAAAAAAAAAA 1632 385 amino acids amino acid single linear protein 2 Met Lys LeuVal Arg Lys Asn Ile Glu Lys Asp Asn Ala Gly Gln Val 1 5 10 15 Thr LeuVal Pro Glu Glu Pro Glu Asp Met Trp His Thr Tyr Asn Leu 20 25 30 Val GlnVal Gly Asp Ser Leu Arg Ala Ser Thr Ile Arg Lys Val Gln 35 40 45 Thr GluSer Ser Thr Gly Ser Val Gly Ser Asn Arg Val Arg Thr Thr 50 55 60 Leu ThrLeu Cys Val Glu Ala Ile Asp Phe Asp Ser Gln Ala Cys Gln 65 70 75 80 LeuArg Val Lys Gly Thr Asn Ile Gln Glu Asn Glu Tyr Val Lys Met 85 90 95 GlyAla Tyr His Thr Ile Glu Leu Glu Pro Asn Arg Gln Phe Thr Leu 100 105 110Ala Lys Lys Gln Trp Asp Ser Val Val Leu Glu Arg Ile Glu Gln Ala 115 120125 Cys Asp Pro Ala Trp Ser Ala Asp Val Ala Ala Val Val Met Gln Glu 130135 140 Gly Leu Ala His Ile Cys Leu Val Thr Pro Ser Met Thr Leu Thr Arg145 150 155 160 Ala Lys Val Glu Val Asn Ile Pro Arg Lys Arg Lys Gly AsnCys Ser 165 170 175 Gln His Asp Arg Ala Leu Glu Arg Phe Tyr Glu Gln ValVal Gln Ala 180 185 190 Ile Gln Arg His Ile His Phe Asp Val Val Lys CysIle Leu Val Ala 195 200 205 Ser Pro Gly Phe Val Arg Glu Gln Phe Cys AspTyr Met Phe Gln Gln 210 215 220 Ala Val Lys Thr Asp Asn Lys Leu Leu LeuGlu Asn Arg Ser Lys Phe 225 230 235 240 Leu Gln Val His Ala Ser Ser GlyHis Lys Tyr Ser Leu Lys Glu Ala 245 250 255 Leu Cys Asp Pro Thr Val AlaSer Arg Leu Ser Asp Thr Lys Ala Ala 260 265 270 Gly Glu Val Lys Ala LeuAsp Asp Phe Tyr Lys Met Leu Gln His Glu 275 280 285 Pro Asp Arg Ala PheTyr Gly Leu Lys Gln Val Glu Lys Ala Asn Glu 290 295 300 Ala Met Ala IleAsp Thr Leu Leu Ile Ser Asp Glu Leu Phe Arg His 305 310 315 320 Gln AspVal Ala Thr Arg Ser Arg Tyr Val Arg Leu Val Asp Ser Val 325 330 335 LysGlu Asn Ala Gly Thr Ala Arg Ile Phe Ser Ser Leu His Val Ser 340 345 350Gly Glu Gln Leu Ser Gln Leu Thr Gly Val Ala Ala Ile Leu Arg Phe 355 360365 Pro Val Pro Glu Leu Ser Asp Gln Glu Gly Asp Ser Ser Ser Glu Glu 370375 380 Asp 385

What is claimed is:
 1. An isolated polypeptide comprising the amino acidsequence set forth in SEQ ID NO:2.
 2. The isolated polypeptide of claim1 consisting of the amino acid sequence set forth in SEQ ID NO:2.
 3. Anantibody immunospecific for the polypeptide of claim 1.